The long term objectives of this research program are to understand the mechanisms by which the cytoskeleton becomes abnormal in Alzheimer's diesease and, perhaps, in normal aged brains. We are particularly interested in the role phosphorylation of cytoskeletal polypeptides might play in mediating the ability of the polypeptides to interact in an appropriate fashion to form noraml cytoskeletal organelles. A major hypothesis we will be testing is that alterations in the state of phosphorylation of cytoskeletal polypeptides in Alzheimer's disease is one step leading to the formation of the paired helical filaments (PHF) in the neurofibrillary tangle (NFT) and neuritic plague (NP) that are a hallmark of the pathology of this disease. Results to date indicate that modifications in the state of phosphorylation of the tau family of microtubule-associated proteins may be a step in the pathogenesis of the NFT and NP. In this program we will use a variety of biochemcial procedures to isolate and purify individual tau isoforms and fragments from AD brains to use in bioassay with regard to their properties in stimulating the assembly and stability of microtubules as a test for those fragments likely to contain biologically relevant modified sites. We will also compare the detailed pattern of tau cytochemistry in buffer and alkaline phosphatase treated NFT and NP at the electron microscopic level to ask whether or not sites accessible to anti-tau without dephosphorylation represent early-forming tangles with relatively unmodified tau associated with some straignt tubules, whereas those sites exposed after dephosphorylation represent tau modified by more extensive phosphorylation to participate in the formation of networks of PHF. We will use phosphorylation by exogenous and endogenous protein kinase activities of tissue sections and membrane blots to further identify and characterize sites on the tau family of polypeptides which might be modified in AD and, potentially, in aged human brains.